Pre-mRNA splicing is the removal of the non-coding introns that interrupt most gene transcripts and serves an essential step in eukaryotic gene expression. Furthermore, splicing is now widely recognized as a key mediator of proteome complexity by regulating alternative inclusion of different exons from the same transcript. It is important to understand the molecular machinery that carries out splicing because mutations that affect both constitutive and alternative splicing are associated with a number of human diseases, including cancers. The machinery, termed the spliceosome, is a large protein/RNA macromolecular complex comprised of five structural RNAs and over 100 individual polypeptides. The spliceosome assembles and functions via a progression of structural intermediates that are not yet fully characterized. The dynamic complexity of the spliceosome has long posed a challenge to detailed biochemical and structural studies. To meet this challenge, small molecule inhibitors that will arrest spliceosomes at different steps along the splicing pathway are needed as tools. There are reports of splicing inhibitors, but their utility as structure-function tools has not yet been realized. Developing an arsenal of potent small molecule inhibitors that target the spliceosome is critical for dissecting its mechanisms and for being able to manipulate its function in disease situations. We propose to develop a complementary set of assays suitable for high-throughput screening (HTS) for potential spliceosome inhibitors. One assay will test splicing in an in vitro setting and simultaneously target all critical spliceosome proteins/interactions. A complementary assay will monitor splicing in vivo in S. cerevisiae and use synthetic lethality with spliceosome mutants to target key steps of spliceosome function. In collaboration with Dr. Scott Lokey in the Chemistry Department at UCSC, we will use the assays to screen several small molecule libraries totaling over 55,000 compounds. Candidate inhibitors identified by either screen will be characterized by established gel-based assays in both human and yeast nuclear extracts to determine at which point of spliceosome dynamic function they exert their effects. Inhibitors identified by these screens will serve as new tools to trap spliceosome for further structural and biochemical studies. The compounds will also serve as lead compounds for developing drugs that target splicing function. PUBLIC HEALTH RELEVANCE: Alterations in the cellular machinery that edits most human gene products (termed spliceosomes) are associated with a number of human diseases, including cancers. Identifying chemicals that block the function of this cellular machinery will provide insights into the workings of spliceosomes and potentially serve as leads for drugs to treat disease.